Abstract This is a proposal for a new, multi-PI grant involving two senior investigators with complementary expertise to study an essential but poorly understood step in human papillomavirus (HPV) infection. HPV is responsible for approximately 5% of human cancer worldwide. During infection, HPV is internalized to the endosome from where it is targeted to the retrograde pathway for transport to the TGN en route to the nucleus for productive infection. Endosome-to-Golgi targeting represents the committed infection step and remains poorly understood. Our published and unpublished results suggest that a host factor ? the transmembrane protease ?-secretase ? is endowed with a novel chaperone activity that promotes insertion of an HPV capsid protein called L2 into the endosome membrane. Membrane insertion of L2 and subsequent protrusion into the cytoplasm recruits cytosolic host components that deliver HPV to the Golgi. Strikingly, we also found that HPV infection stabilizes the multi-subunit ?-secretase complex. Accordingly, the central objectives of this proposal are to elucidate, at the molecular level, how ?- secretase promotes membrane insertion of L2 and how HPV regulates the function of ?-secretase to coordinately promote HPV infection. We will identify cellular proteins that recruit ?-secretase to HPV, determine if ?-secretase-mediated capsid rearrangements allow L2 to emerge from the capsid, and attempt to reconstitute membrane insertion in vitro and determine the relevant ?-secretase subunits and activities. We will determine if ?-secretase plays a role in insertion of cell-penetrating peptides or retrograde trafficking in general. We will examine the role of the N-terminal transmembrane domain in L2 in these activities and attempt to develop viral mutants that are independent of ?-secretase. Finally, we will determine how HPV infection stabilizes the ?-secretase complex, identify other factors involved in this process, and determine if the L2 transmembrane domain is sufficient for stabilization and membrane insertion. Taken together, these experiments will shed new light on the unusual role of ?-secretase in HPV entry, improve our understanding of this crucial step in HPV infection, elucidate new aspects of general cell biology, and possibly point the way to new therapeutic approaches to prevent infection by these important human pathogens.